The aim of human proteomics is to identify all the proteins of the human body, to determine their ever-changing structures and modifications, to identify their interaction partners and to find out the partners and type of interactions with other proteins. The 22,000 genes which have been found in the human genome generate a much larger number of different types of proteins (100 to 1000 times more) as a result of mutations, posttransscriptional and posttranslational modifications of the proteins. The task of human proteomics is therefore immense. Further tasks are waiting in animal proteomics and plant proteomics. In the end, success in researching these building blocks of our life will depend on the development of effective tools for identifying protein structures and their modifications.
Tandem mass spectrometry (MS/MS) is one of most useful tools in proteomics because of its high detection power (femtomoles and lower) and its high specificity. The conventional MS/MS method of protein characterization consists in enzymatic digestion of the protein and subsequent fragmentation of the digest peptides in the mass spectrometer by collisions with a collision gas. (Definition: peptides are small proteins with up to about 30 or 40 amino acids; digest peptides are formed from larger proteins by enzymatic digestion, for example by trypsin). The masses of the fragment ions and the molecular ion are then entered into a search engine, which compares the measured fragmentation pattern with theoretical fragmentation patterns of the virtual digest peptides of all the proteins in a protein sequence database. The success of this method depends on how many fragment ions are formed from the digest peptide ion and how characteristic these fragment ions of a given digest peptide are.
The customary fragmentation technique is collisionally induced fragmentation (CID=collision induced dissociation, also often called CAD=collisionally activated dissociation). The peptide ions are accelerated to kinetic energies of between 20 and 4000 electronvolts and collide with molecules of neutral gas, thereby exciting internal bonding systems to oscillations. CID preferably splits the so-called peptide bonds (C—N bonds in the central chain of amino acids) thereby forming so-called N-terminal B fragments and C-terminal Y fragments. The disadvantages of this fragmentation are, firstly, losses of side chains which are easily split off—these groups occur in many posttranslational modifications (for example phosphorylations and sulfations)—and, secondly, the incomplete fragmentations which frequently occur.
In reality, the information on the protein transmitted to the database search engines in the form of MS/MS spectra is rarely complete, and therefore false identifications cannot be ruled out. In fact, they occur rather frequently because the databases contain only a minute fraction of all actually occurring proteins. Even if the genome has been completely decoded, particularly all mutational forms and modifications are lacking in the data base. False identifications and incorrect structural information are a serious problem of present day proteomics.
To avoid false identifications and incorrect structural information, independent (so-called “orthogonal”) and preferably also gentle types of fragmentation must be available. Methods which are orthogonal to each other provide confirmatory information via other, independent means. These orthogonal fragmentation methods can be drawn on to confirm the identifications and correct the structural information. A good candidate for a type of fragmentation orthogonal to collisionally induced fragmentation CID is electron capture dissociation (ECD), which splits N—Ca bonds of the amino acid chain and generates N-terminal C fragments and C-terminal Z fragments without losing labile groups in the process. The mass difference between B and C ions of the same type is +17 atomic mass units (Daltons), and the difference between Z and Y ions is −16 Daltons. These mass differences, which must occur between the independent measurements, given the correct identification, can make the identification more certain. Applying a combination of CID and ECD increases the certainty of the identification by factors of between 20 and 100.
Up to now, fragmentation by electron capture ECD could only be used routinely in expensive ion cyclotron resonance mass spectrometers ICR-MS (also called Fourier transform mass spectrometer FTMS). The prerequisite for this fragmentation method is that doubly charged ions of the analytical substance (termed analyte substance below) are available.
A further method of fragmentation has recently been published which also is orthogonal to collisionally induced fragmentation CID: The fragmentation of multiply charged positive ions by reactions with suitable negative ions by electron transfer is called “electron transfer dissociation” or ETD, described by J. E. P. Syka, J. J. Coon, M. J. Schroeder, J. Shabanowitz and D. F., Hunt in the paper “Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry”, Proc. Natl. Acad. Sci. USA 2004, 101, 9528-9533. These reactions can take place in RF ion traps, which means both linear ion traps made of rod electrodes and three-dimensional ion traps made of ring electrodes and end cap electrodes. But to obtain high yields of fragment ions, it is necessary to start with triply charged ions. For this reason the method has its limitations, because precisely for digest peptides—which are by far the most interesting biomolecules—triply charged ions can only be produced with a very limited yield. The fact that it is necessary to begin with triply charged analyte ions reduces the detection power of the method with ETD by factors of between 10 and 20.
A commonly used way of ionizing large biomolecules is to use electrospray ionization (ESI), which ionizes ions at atmospheric pressure outside the mass spectrometer. These ions are then introduced into the vacuum of the mass spectrometer, and from there into the ion trap by means of inlet systems of a type which has already been described.
This type of ionization generates hardly any fragment ions. The ions are mostly those of the analyte molecule. Electrospray ionization does, however, produce multiply charged analyte ions. In the case of peptides, the doubly charged ions are the most prevalent ions in around 85 to 90 percent of cases. The triply charged ions generally represent only a few percent of all the ions formed. For lighter peptides, the singly charged ions are the most prevalent, the doubly charged ones the second most prevalent. The lack of almost any fragmentation of the analyte ions created during the ionization process limits the information from the mass spectrum to the molecular weight; there is no information concerning internal molecular structures which can be used for further identification of the substance present. This information can only be obtained by the above-described acquisition of fragment ion spectra in tandem mass spectrometers.
Owing to the high prices of Fourier transform mass spectrometers (FTMS) it would be desirable for fragmentation methods which are complementary and orthogonal to collisionally induced fragmentation CID, such as electron capture dissociation ECD, to also be carried out in simpler, smaller and less expensive mass spectrometers, for example in quadrupole ion trap mass spectrometers operated with RF voltages. Until now, the fragmentation in quadrupole ion traps has been exclusively carried out using collisionally induced fragmentation CID. For fragmentation by electron capture ECD, on the other hand, the kinetic energy of the electrons must be very low, as otherwise no capture can take place. In practice one supplies electrons with an energy just above the thermal energy. This works very well in the very strong magnetic fields of the Fourier transform mass spectrometer, but not in electric RF ion traps. It has not yet proven possible, neither in three-dimensional nor in linear ion traps, to fragment the ions by electron capture and obtain a high enough yield. The disrupting factor here is the fact that there is practically always a high electric field strength, which makes it difficult, if not impossible, for low-energy electrons to enter (in spite of patents in this fields, see J. Franzen DE 100 58 706 C1; or R. Zubarev et al. U.S. Pat. No. 6,800,851 B1).
Ion traps according to Wolfgang Paul normally comprise a ring electrode and two end cap electrodes, the ring electrode usually being supplied with the storage RF voltage. These ion traps are also called three-dimensional ion traps (“3D ion traps”). It is also possible to use four-rod quadrupole filters according to Paul as ion traps if both ends of the rod system are supplied with ion-repelling potentials through diaphragms. These so-called “linear quadrupole ion traps”, or “linear ion traps” for short, are easier to fill with ions, and with slightly more ions than the “three-dimensional ion traps”. In the interior of the ion trap, ions can be stored in the quadrupole RF field. Linear ion traps are also often called “two-dimensional ion traps” or “2D ion traps”.
Both three-dimensional and linear ion traps can also be employed as ion analyzers by using resonant excitation to eject the ions selectively according to mass and then measuring them as ion currents. The ions can be mass-selectively ejected from the linear quadrupole ion traps either radially through slits in at least one of the long electrodes (U.S. Pat. No. 5,420,425, M. E. Bier and J. E. Syka, which corresponds to EP 0 684 628 A1), or axially by means of coupling processes in the inhomogeneous end field of the rod system (“A new linear ion trap mass spectrometer”, J. W. Hager, Rapid Commun. Mass Spectrom. 2002, 16, 512-526). The mass-selectively ejected ions are measured by a detection unit, for example a secondary-electron multiplier, and then the measurements can be processed to a mass spectrum.
Ion trap mass spectrometers have properties which make them of interest for use in many types of analyses. In particular, selected ion species (so-called “parent ions”) can be isolated and fragmented in the ion trap. The isolation consists in ejecting all undesired ions from the ion trap by resonant excitation and only leaving the desired analyte ions in the ion trap. The fragmentation occurs in a slightly different way to the fragmentation by acceleration of the analyte ions described above. By exciting their oscillations, the analyte ions are forced to undergo a large number of individual collisions with the collision gas, thereby absorbing very small portions of energy until finally a fragmentation occurs. Both this type of fragmentation and the collisionally induced fragmentation after acceleration provide fragment ions of the B and Y series. The spectra of these fragment ions are also called “daughter ion spectra” of the respective parent ions. In ion traps, “granddaughter ion spectra” can also be measured as fragment ion spectra of selected daughter ions.